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Phosphorylation and Action of the Immunomodulator FTY720 Inhibits Vascular Endothelial Cell Growth Factor-induced Vascular Permeability

2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês

10.1074/jbc.m306896200

ISSN

1083-351X

Autores

Teresa Sánchez, Tatiana Estrada-Hernandez, Jihye Paik, Ming-Tao Wu, Krishnan Venkataraman, Volker Brinkmann, Kevin P. Claffey, Timothy Hla,

Tópico(s)

Platelet Disorders and Treatments

Resumo

FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for sphingosine 1-phosphate (S1P) receptors. The effects of FTY720 on vascular cells, a major target of S1P action, have not been addressed. We now report the metabolic activation of FTY720 by sphingosine kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by sphingosine kinase activity and formation of FTY720-P. Sphingosine kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for Gi-coupled S1P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth. FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for sphingosine 1-phosphate (S1P) receptors. The effects of FTY720 on vascular cells, a major target of S1P action, have not been addressed. We now report the metabolic activation of FTY720 by sphingosine kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by sphingosine kinase activity and formation of FTY720-P. Sphingosine kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for Gi-coupled S1P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth. Sphingosine-1-phosphate (S1P), 1The abbreviations used are: S1Psphingosine 1-phosphateSKsphingosine kinaseEDGendothelial differentiation geneERKextracellular signal-activated kinaseVEGFvascular endothelial cell growth factorDMSN,N′-dimethyl sphingosineFITCfluorescein isothiocyanateBSAbovine serum albuminHUVEChuman umbilical vein endothelial cell(s)RTreverse transcriptionP-phosphorylatedVEvascular endothelial. a multifunctional bioactive lipid mediator, mediates cellular responses by activating G protein-coupled receptors (originally termed as EDG receptors but that has been renamed as S1Pn receptors). To date, five S1P receptors have been identified in different cell types, S1P1/EDG-1, S1P2/EDG-5, S1P3/EDG-3, S1P4/EDG-6, and S1P5/EDG-8 (1Lee M.J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (890) Google Scholar, 2Ancellin N. Hla T. J. Biol. Chem. 1999; 274: 18997-19002Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 3Van Brocklyn J.R. Tu Z. Edsall L.C. Schmidt R.R. Spiegel S. J. Biol. Chem. 1999; 274: 4626-4632Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 4Van Brocklyn J.R. Graler M.H. Bernhardt G. Hobson J.P. Lipp M. Spiegel S. Blood. 2000; 95: 2624-2629Crossref PubMed Google Scholar, 5Im D.S. Heise C.E. Ancellin N. O'Dowd B.F. Shei G.J. Heavens R.P. Rigby M.R. Hla T. Mandala S. McAllister G. George S.R. Lynch K.R. J. Biol. Chem. 2000; 275: 14281-14286Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 6Hla T. Lee M.J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (482) Google Scholar, 7Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar). Interaction of S1P with these receptors regulates a multitude of effects in the vascular system, including endothelial cell proliferation, survival, adherens junction assembly, migration, and morphogenesis (6Hla T. Lee M.J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (482) Google Scholar, 7Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar). In particular, activation of S1P1 and S1P3 receptors plays an important role in survival and morphogenesis in endothelial cells in vitro (8Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar), and S1P1 is necessary for vascular maturation in vivo (9Liu Y. Wada R. Yamashita T. Mi Y. Deng C.X. Hobson J.P. Rosenfeldt H.M. Nava V.E. Chae S.S. Lee M.J. Liu C.H. Hla T. Spiegel S. Proia R.L. J. Clin. Invest. 2000; 106: 951-961Crossref PubMed Scopus (996) Google Scholar). However, S1P also exerts potent effects on other organ systems, such as the immune system and the reproductive system among others, indicating that it is a multifunctional lipid mediator (6Hla T. Lee M.J. Ancellin N. Paik J.H. Kluk M.J. Science. 2001; 294: 1875-1878Crossref PubMed Scopus (482) Google Scholar, 7Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar, 10Huang M.C. Graeler M. Shankar G. Spencer J. Goetzl E.J. Biochim. Biophys. Acta. 2002; 1582: 161-167Crossref PubMed Scopus (62) Google Scholar). sphingosine 1-phosphate sphingosine kinase endothelial differentiation gene extracellular signal-activated kinase vascular endothelial cell growth factor N,N′-dimethyl sphingosine fluorescein isothiocyanate bovine serum albumin human umbilical vein endothelial cell(s) reverse transcription phosphorylated vascular endothelial. S1P is generated by the metabolism of sphingomyelin, an abundant component of biological membranes. Specifically, sphingomyelinase degrades sphingomyelin to ceramide, which is degraded by a ceramidase to sphingosine, the substrate for sphingosine kinase (SK) (11Spiegel S. Merrill Jr., A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (650) Google Scholar), which adds the phosphoryl group to the 1-hydroxyl group of the sphingosine molecule. S1P can be converted to sphingosine by the S1P-phosphohydrolase (12Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 13Le Stunff H. Peterson C. Thornton R. Milstien S. Mandala S.M. Spiegel S. J. Biol. Chem. 2002; 277: 8920-8927Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) or degraded irreversibly by S1P-lyase (14Saba J.D. Nara F. Bielawska A. Garrett S. Hannun Y.A. J. Biol. Chem. 1997; 272: 26087-26090Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Most cells exhibit a basal tone of SK activity that is further increased by different extracellular stimuli, such as growth factors, cytokines, and hormones. For example, platelet-derived growth factor (15Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (818) Google Scholar), tumor necrosis factor (16Xia P. Gamble J.R. Rye K.A. Wang L. Hii C.S. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci. U. S. 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Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 22Nikolova-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 18718-18724Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and sphingomyelinase (23Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 24Wong M.L. Xie B. Beatini N. Phu P. Marathe S. Johns A. Gold P.W. Hirsch E. Williams K.J. Licinio J. Tabas I. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8681-8686Crossref PubMed Scopus (139) Google Scholar). These studies suggest that agonist-induced formation of S1P may serve a regulatory role in cellular signaling. The central enzyme in the pathway, SK, is encoded by two SK genes encoding SK1 and SK2 isoenzymes (25Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog. Nucleic Acid Res. Mol. Biol. 2002; 71: 493-511Crossref PubMed Google Scholar). However, the physiological role of each of these kinases remains to be elucidated. Platelets, which exhibit high SK activity and lack the S1P lyase, are a rich source of S1P in vivo (26Yatomi Y. Ruan F. Hakomori S. Igarashi Y. Blood. 1995; 86: 193-202Crossref PubMed Google Scholar). Platelets contain stored S1P (27Yatomi Y. Yamamura S. Ruan F. Igarashi Y. J. Biol. Chem. 1997; 272: 5291-5297Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and release it upon activation, thus contributing to serum S1P levels. Moreover, neutrophils, erythrocytes, endothelial cells, and mononuclear cells also release S1P (28Yang L. Yatomi Y. Miura Y. Satoh K. Ozaki Y. Br. J. Haematol. 1999; 107: 282-293Crossref PubMed Scopus (112) Google Scholar, 29Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S.S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). However, the mechanisms whereby S1P is released are poorly understood. Studies from our laboratory recently showed that endothelial cells actively export the SK1 enzyme into the extracellular milieu, which could explain some of the biological actions of S1P (29Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S.S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Thus, intracellular and extracellular S1P-generating systems exist and contribute to circulating S1P levels. A novel pharmacologic modulator of S1P receptors has been described, namely, the immunomodulatory agent, FTY720 (30Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1443) Google Scholar, 31Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar). FTY720 elicits lymphopenia in blood and thoracic duct by sequestration of lymphocytes from circulation to secondary lymphoid organs, away from inflamed peripheral tissues and graft sites. FTY720 is phosphorylated in vivo, and the phosphorylated form (FTY720-P) is a potent agonist of S1P1, S1P3, S1P4, and S1P5 receptors (30Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1443) Google Scholar, 31Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar). Similarly, the analog (R)AAL (but not the chiral analog (S)-AAL), was also phosphorylated into (R)-AFD (31Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar). These important findings provide a critical clue for the mechanism of action of this potent immunomodulatory agent. However, the cellular and molecular basis of FTY720 action remains to be elucidated. The vascular system contributes greatly to the innate as well as adaptive immunity and in fact plays a critical role in transplant-associated tissue rejection (32Biedermann B.C. Sahner S. Gregor M. Tsakiris D.A. Jeanneret C. Pober J.S. Gratwohl A. Lancet. 2002; 359: 2078-2083Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). In this report, we show that FTY720 possesses a heretofore unappreciated function as a profound regulator of the vasculature in vivo. Reagents—Fatty acid-free bovine serum albumin (BSA), 4-deoxypyridoxine, β-glycerophosphate, and fluorescein isothiocyanate (FITC)-dextran were purchased from Sigma. Sphingosine, N,N-dimethylsphingosine (DMS), and S1P were purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). [γ-32P]ATP (specific activity 6,000 Ci/mmol) and [methyl-3H]thymidine (86 Ci/mmol) were from Amersham Biosciences. FTY720, phospho FTY720, (S)-AAL, (R)-AAL, and phospho-(R)-AAL were kindly provided by Dr. V. Brinkmann, Novartis. Pertussis toxin was purchased from Calbiochem. Phospho-Akt, Akt, phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), and ERK-1/2 antibodies were from Cell Signaling Tech., and VE-cadherin and β-catenin antibodies were from Santa Cruz Biotechnology. Cloning of SK1 and SK2—Mouse liver RNA (Ambion) was used to amplify the murine SK1 and SK2 cDNA by reverse transcriptase (RT) PCR with the forward primers 5′-AGC CCC ATG TGG TGG TGT TGT-3′ and 5′-ATT ATG GCC CCA CCA CCA CTA CT-3′, respectively and reverse primers 5′-GGC ACA GAG TTA TGG TTC TTC-3′ and 5′-AGG TCA GGC TTG TGG CTT TTG AC-3′, respectively. The resulting PCR product was cloned in pcDNA3.1 Topo vector (Invitrogen), and the DNA sequence was confirmed. SK1 and SK2 cDNAs were then cloned in pcDNA3.1 eukaryotic expression vector (Invitrogen). Full-length cDNA clones for human SK1 (IMAGE_ID 3831657; BC008040) and SK2(IMAGE_ID; BC006161) were procured from Protein Tech Group, Chicago, IL. The SK1 and SK2 inserts were released by EcoRI and XhoI digestion, and the resulting 1.8- and 3.0-kbp fragments, corresponding to SK1 and SK2, were subcloned into pcDNA 3.0 mammalian expression vector at EcoR1 and XhoI sites. Reverse Transcription and Real Time PCR—Total RNA was isolated from cells using the RNA-Stat 60 reagent (Tel Test). 1 μg of total RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI) and reverse-transcribed using random hexamer primers (Invitrogen) and Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacture's instructions. Primers were designed using Primer Express™ 2.0 (Applied Biosystems) according to the software guidelines. All primer pairs were designed for a melting temperature of 60 °C. Primer sequences were as follows: human glyceraldehyde-3-phosphate dehydrogenase forward (5′-TGC ACC ACC AAC TGC TTA GC-3′) and reverse (5′-GGC ATG GAC TGT GGT CAT GAG-3′), human SK1 forward (5′-CATCCAGAAGCCCCTGTGTAG-3′) and reverse (5′-GTC TTC ATT GGT GAC CTG CTC AT-3′), human SK2 forward (5′-CAA CCT CAT CCA GAC AGA ACG A-3′) and reverse (5′-TTC ACA GCT TCC TCC CAG TCA-3′), mouse SK1 forward (5′-GGA GGA GGC AGA GAT AAC CTT TAA A-3′) and reverse (5′-GAC CCA ACT CCT CTG CAC ACA-3′), mouse SK2 forward (5′-GTG GTG CCA ATG ATC TCT GAA G-3′) and reverse (5′-GCTCACGGGCATGGTTCT-3′). Real time PCR was performed using QuantiTect SYBR Green PCR kit (Qiagen) on an ABI Prism 7900 HT Sequence Detection System (PE Applied Biosystems, Foster City, CA). Each reaction was run in duplicate and contained 5 μl of cDNA, 900 nm primers and 12.5 μl of SYBR Green PCR master mix in a 25-μl final volume. The parameters for PCR were 95 °C for 15 s and 60 °C for 1 min for 40 cycles and a final dissociation stage of 95 °C for 15 s. Melting curves were performed using the SDS2.1 software (Applied Biosystems) to ensure that only a single product of the correct melting temperature was amplified. Quantification of RNA levels was calculated as previously described (33Liu W. Saint D.A. Biochem. Biophys. Res. Commun. 2002; 294: 347-353Crossref PubMed Scopus (293) Google Scholar). Cell Culture and Transfection—Human umbilical vein endothelial cells (HUVEC) (Clonetics, p4–11) were cultured in M199 medium supplemented with 10% fetal bovine serum and heparin-stabilized endothelial cell growth factor, as previously described (34Hla T. Maciag T. J. Biol. Chem. 1990; 265: 9308-9313Abstract Full Text PDF PubMed Google Scholar). Human embryonic kidney 293T cells in 60-mm dishes were transfected with 4 μg of vector alone or with vectors containing SK constructs by the calcium phosphate method. 24 h after transfection, cells were harvested by scraping at 4 °C with 400 μl of 25 mm HEPES, pH 7.5, 5 mm MgCl2,1× protease inhibitor mixture (Calbiochem), and disrupted by brief sonication. Cellular homogenates were centrifuged for 10 min, 4 °C at 20,000 × g. SK activity was determined as described under "Materials and Methods." Cell Migration Assay—HUVEC migration was assayed using a 96-well chemotaxis microchamber (Neuro Probe, Inc.) as previously described (35Paik J.H. Chae S. Lee M.J. Thangada S. Hla T. J. Biol. Chem. 2001; 276: 11830-11837Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Briefly, polycarbonate filters with a pore size of 8 μm were coated with 5 μg/ml fibronectin. S1P, FTY720, AAL, FTY720-P, or (R)-AFD was diluted in 0.1% fatty acid free BSA into the appropriate concentration. 85 μl of the solutions or conditioned medium from HUVEC were added into the lower chamber. Approximately 5 × 104 cells suspended in M199 with 0.1% bovine serum albumin were placed in the upper compartment. The cells were allowed to migrate for 4 h at 37 °C in a humidified chamber with 5% CO2. After the incubation period the filter was removed, and cells were stained in 0.1% crystal violet and eluted with 10% acetic acid in 96-well plates. Quantification was done based on absorbance at 575 nm by a Spectramax 340 (Molecular Devices) plate reader. Activation of FTY720 and (R)-AAL by Endothelial Cells—Cells were washed 3 times with plain medium M199 and incubated with 0.1% fatty acid free BSA containing different concentrations of FTY720 or (R)-AAL for 3 h. After incubation, conditioned medium was removed, centrifuged at 1000 × g for 10 min, and used in the migration assay. Analysis of Kinase Activity from Conditioned Media and Cells—Cells were washed 3 times with plain medium M199 and incubated with 0.1% fatty acid free BSA for the times indicated. After incubation, conditioned medium was removed, centrifuged at 1,000 × g for 10 min, and used for the kinase activity assay. Cells were scraped at 4 °C with 400 μl of 25 mm HEPES, pH 7.5, 5 mm MgCl2, 1× protease inhibitor mixture, and disrupted by brief sonication. Cellular homogenates were centrifuged for 10 min, 4 °C at 20,000 × g. SK activity was determined as described under "Materials and Methods." In Vitro Kinase Assay—300 μl of conditioned medium (derived from 5 × 105cells for 3 h of incubation) or 10 μg of total cell extract from HUVEC were used as a source of kinase activity. Reactions contained 20 μm sphingosine, 100 μm FTY720, or AAL, [γ-32P]ATP (10 μCi), 5 mm MgCl2, 15 mm NaF, 0.5 mm 4-deoxypyridoxine, 40 mm β-glycerophosphate, and 300 μl of M199 and were incubated at 37 °C for 30 min. Lipids were extracted as previously described (29Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S.S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), and samples were resuspended in 50 μl of chloroform. Lipids were resolved by TLC on silica gel G60 using 1-butanol/acetic acid/water (60/20/20 v/v) buffer and quantified with a PhosphorImager (Molecular Dynamics). Western Blot Analysis—Confluent cultures of HUVEC cells were serum-starved for 2 h in 0.1% fatty acid-free BSA M199 before treatments. Then they were washed with ice-cold phosphate-buffered saline containing 1 mm sodium fluoride and 1 mm sodium orthovanadate and homogenized in radioimmunoprecipitation assay buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 1 mm sodium orthovana-date, 50 mm β-glycerophosphate, and 1× protease inhibitor mixture). Samples were centrifuged 10,000 × g for 10 min, and protein concentrations of supernatants were determined by Bradford assay (Bio-Rad protein dye reagent). Equal amounts of protein were separated on a 10% polyacrylamide gel and blotted to a nitrocellulose membrane. Immunoblot analysis was performed using phospho-Akt, Akt, phospho-ERK1/2. or ERK-1/2 antibodies. Apoptosis—HUVEC cells were labeled with [methyl-3H]thymidine (1 μCi/ml) for 16 h. Then they were washed, and 0.1% BSA M199 containing the different treatments was added. After 8 h, fragmented DNA was solubilized and quantified as described (36Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1357) Google Scholar). Immunofluorescence Analysis—2 × 105 cells were plated in 35-mm glass-bottom Petri dishes. Three days later, cells were washed and serum-starved for 3 h before the experiment. Then cells were washed with ice-cold phosphate-buffered saline and fixed, and immunofluorescence analysis with VE-cadherin and β-catenin antibody (1 μg/ml; Santa Cruz) was conducted. Antibody staining was visualized with Alexa Fluor 488 donkey anti-goat (1:1000) IgG (Molecular Probes). Confocal microscopy was conducted on a Zeiss LSM 510 laser-scanning confocal microscope at the Center for Biomedical Imaging at the University of Connecticut Health Center. Fluorescence was excited using a 488-nm argon laser, and emitted fluorescence was detected with a 505-nm long-pass filter. VEGF-induced Trans-cellular Permeability in Vitro—Mouse embryonic endothelial cells (5 × 104) were cultured for 2 days in Transwell polycarbonate filters (6.5-mm diameter, 0.4-μm pores; Costar). Culture medium was replaced with serum/phenol red-free Dulbecco's modified Eagle's medium (0.1-ml upper chamber and 0.6-ml lower chamber). Cells were pretreated with FTY720, derivatives, or S1P for 1 h. FITC-dextran (average Mr = 2000 kDa) was added to the upper compartment either in the absence or presence of murine recombinant VEGF (50 ng/ml). Media from lower wells were taken after the indicated time periods (5–45 min). Samples were placed in black solid-bottom 96-well plates (Costar), and fluorescence intensities were measured using a CytoFluor(R) fluorescence multi-well plate reader (Applied Biosystems) at 488-nm excitation. VEGF-induced Microvascular Permeability—Normal FVB/N female or male mice were treated by gavage with FTY720 (50 μg), (S)-AAL enantiomer (50 μg), (R)-AAL enantiomer (50 μg) and water as the vehicle control. Five hours post-gavage animals were anesthetized with 2% Avertin™ (0.5 cc/20 g) and infused through the tail vein with 100 μl of FITC-dextran (5 mg/ml, 165 kDa). Animals were placed on a warming table under a fluorescent dissecting microscope (Zeiss STV11, Zeiss, Inc.), and the central vessels in the ear were imaged. Control saline or mouse vascular endothelial growth factor (VEGF, 10 ng/ml) was injected subdermally into the middle ear using a 30-gauge needle (30 μl). Ear vasculature was then imaged with fixed exposure times in a stationary position from 5 to 120 min post-injection. Fluorescence images were then quantified using a pixel-based threshold in ImageProPlus (Media Cybernetics, Inc.) and quantified. Transmission Electron Microscopy of the Vasculature—Animals pretreated by gavage with water, FTY, or (S)-AAL for 5 h were used to inoculate the ear subcutaneously with mouse VEGF (10 ng/ml, 50 μl) while under anesthesia. Five minutes post-injection, the ears were removed and fixed in 2% glutaraldehyde/sodium cacodylate buffer, trimmed to isolate the injection site, and processed for osmium tetroxide post-fixation. After post-fixation, tissues were stained with uranyl acetate (0.5% in H2O), serially dehydrated with ethanol, and embedded in Polybed (Polysciences, Warning, PA). Analysis of the cross-section was performed by transmission electron microscopy on a Philips CM10. Sections (70–90-nm thick) were obtained, and vessels adjacent to the ear cartilage were photographed at various magnifications (8Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar). Statistical Analysis—All experiments were performed two to five times, and a representative experiment is shown. In migration and apoptosis experiments results represent mean values of triplicates. p values were calculated by Student's t test using Microsoft Excel software. Phosphorylation of FTY720 by Sphingosine Kinases in Vascular Endothelial Cells—Because S1P is a potent inducer of endothelial cell chemotaxis, we tested the effect of FTY720 and its analogues on endothelial cell migration. Neither FTY720 nor its structural analog (R)-AAL (Fig. 1a) induced endothelial cell migration in a wide concentration range (Fig. 1b). However, when FTY720 or (R)-AAL was incubated with HUVEC for 3 h, they were activated into potent chemoattractants for endothelial cells. This conditioned medium-induced migration was dose-dependent and pertussis toxin-sensitive, indicating the involvement of a Gi-coupled receptor. In contrast, the enantiomer (S)-AAL, which cannot be phosphorylated by SK (31Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1320) Google Scholar), did not induce migration after incubation with endothelial cells. These data are consistent with the notion that endothelial cells phosphorylate and activate FTY720 and (R)-AAL into FTY720-P and (R)-AFD, respectively, which are both potent chemoattractants for endothelial cells. Indeed we detected the presence of the SK1 and -2 transcripts in HUVEC cells by RT-PCR (Fig. 1c). We also tested the effect of DMS, which is a competitive inhibitor of the SK enzyme (25Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog. Nucleic Acid Res. Mol. Biol. 2002; 71: 493-511Crossref PubMed Google Scholar, 37Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 38Nava V.E. Lacana E. Poulton S. Liu H. Sugiura M. Kono K. Milstien S. Kohama T. Spiegel S. FEBS Lett. 2000; 473: 81-84Crossref PubMed Scopus (89) Google Scholar). DMS treatment (10 μm) potently inhibited the ability of endothelial cells to activate FTY720 and (R)-AAL (Fig. 1d). These results suggest that the endothelial cell-derived SK phosphorylates and activates both FTY720 and (R)-AAL in a stereospecific manner. To confirm that FTY720 and (R)-AAL are indeed phosphorylated by endothelial cells, we performed an in vitro kinase assay with both total cell extract and conditioned medium from HUVEC. FTY720 and (R)-AAL were phosphorylated by the kinase activity present in HUVEC cytosol (Fig. 2a, top panel, specific activity 3.85 ± 0.75 and 15 ± 0.93 pmol/mg/min, respectively). As expected, sphingosine was an efficient substrate for this kinase activity (specific activity 75.7 ± 6 pmol/mg/min). In sharp contrast, (S)-AAL phosphorylation was undetectable, in agreement with the results obtained with the migration assay. Phosphorylation of both FTY720 and (R)-AAL was inhibited by DMS (50 μm). Similarly, we also detected this kinase activity in the conditioned medium from HUVEC, although this activity was much lower than the cytosolic extracts (Fig. 2a, bottom panel), suggesting that both intracellular and extracellular SK enzyme systems are capable of phosphorylating FTY720 and its analog, (R)-AAL. Interestingly, this phosphorylation was inhibited by Triton X-100 and increased by KCl (200 mm) (data not shown), which are conditions that favor the enzymatic activity of the SK2 isoenzyme (25Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog.

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